KR20110057365A - Method for the production of the transgenic chicken containing the human lactoferrin gene - Google Patents

Abstract

PURPOSE: A method for producing a transformed chicken containing human lactoferrin gene is provided to effectively produce a target proteins. CONSTITUTION: A lentivirus vector contains a promoter region in which human lactoferrin gene and ovalbumin gene are combined together. A recombinant lentivirus is prepared using the lentivirus vector in virus packaging cell, 293T cells. A method for producing a transformed chicken with human lactoferrin gene: a step of analyzing semen from transformed roosters and selecting semen containing human lactoferrin gene; and a step of fertilizing semen with hen and hatching.

Description

Lentivirus vector combining human lactoferrin gene and promoter region of chicken ovalbumin gene, and method for producing chicken having human lactoferrin gene using the same {Method for the production of the transgenic chicken containing the human lactoferrin gene}

The present invention relates to a method for producing a transgenic chicken comprising a human lactoferrin gene, and more particularly, to a recombinant lenti using a lentiviral vector in which a human lactoferrin gene and a promoter region of a 2 kb ovalbumin gene of chicken are combined. The present invention relates to a method for producing a chicken having a human lactoferrin gene after preparing a virus and artificially incubating a fertilized egg using the same.

Advances in modern science and technology, especially in the medical sector, have led to an increase in the lifespan of humans, which has led to an increasing demand for bioactive proteins useful for humans. Currently, the most common bioactive protein production method is Escherichia coli, but the production method using Escherichia coli cannot produce a protein having a complex structure in which sugar is bound. Therefore, a complex structure protein is produced using an animal cell line. . Recently, some researches have been made to produce useful proteins using livestock, and some have reported visible results. To date, most of the animals that are used to produce bioactive protein are mainly used in the secretion gland (medium) of medium and large animals.

Considering these circumstances, chicken eggs are considered to be of sufficient value as model animals for the production of useful proteins. However, unlike mammals, chickens have a problem in that the production efficiency of transgenic chickens is very low because the eggs are one fertilized egg, but chickens have a higher number of litters and shorter generation intervals than mammalian livestock. Mass breeding is possible. Laying hens in particular produce 300-330 eggs per year.

Due to the fact that the egg is a fertilized egg, it is relatively difficult to handle, and the nucleus in the embryo of the fertilized egg cannot be visually observed with a microscope. not.

Transformation methods of chickens developed to date can be divided into recombinant viruses and expression vectors using foreign gene transport methods, and primordial depending on the type of fertilized egg or cell receiving the foreign gene. germ cell), 1-cell fertilized egg and blastocyst stage embryo (stage-X). Until now, the most common method of producing transgenic chickens has been reported to be the most efficient method of producing transgenic chickens by microinjecting recombinant viruses into embryonic stage embryos.

In the present invention, a human lactoferrin gene is introduced by microinjecting a recombinant lentivirus having a human lactoferrin gene into an ovalbumin promoter of a chicken in a stage-X fertilized egg of a chicken. It is to provide a method for producing a transgenic chicken.

In order to solve the above problems, the present invention provides a lentiviral vector in which a promoter region of human lactoferrin gene and ovalbumin gene of 2 kb size is combined with chicken.

In addition, the present invention provides a recombinant lentiviral produced by 293FT virus packaging cells using a lentiviral vector.

In addition, in the artificial culture method of chicken fertilized eggs, the lactoferrin of human characterized in that it comprises the step of culturing the chicken eggs and then moving the contents to a surrogate egg, and injecting a recombinant lentivirus to the transferred contents Provided is an artificial culture method for chicken fertilized eggs having a gene.

In addition, the semen of the transgenic chickens is analyzed to select the presence of human lactoferrin gene, and the selected male semen is artificially inseminated by hatching by insemination of the transgenic chicken. Provides a method of producing converted chicken.

As described above, the present invention has developed a method for producing a second generation transgenic chicken (G1) in which a gene in which a human lactoferrin gene is bound to a chicken ovalbumin promoter is introduced. The present invention provides the applicability of chickens as one bioreactor to produce proteins useful for humans.

1. Recombinant viral vector construction and virus production

The present invention was intended to construct a lentiviral vector in which the promoter region of the lactoferrin gene of human and the ovalbumin gene of 2 kb size in chicken is combined. First, the ovalbumin promoter region and human lactoferrin gene of chicken were amplified by PCR method to obtain DNA fragments of approximately 2.1 and 2.0 kb, respectively (FIG. 2). The amplified genes were constructed using a lentiviral vector as shown in FIG. 3. The constructed viral vector was confirmed to correctly clone the promoter region and human lactoferrin gene to be cloned through sequencing (FIG. 4). In addition, the cloned human lactoferrin gene confirmed that codons existed at the beginning (ATG) and end (TAA) of protein synthesis.

Recombinant lentivirus was produced in 293FT, a virus packaging cell, using a lentivirus vector carrying a human lactoferrin gene. The titer of the recombinant lentivirus produced was quantified by Western blot analysis of the amount of gag protein, a recombinant virus surface protein.

2. Microinjection and artificial fertilization of fertilized eggs

The blastocyst stage fertilized egg according to the present invention used a fertilized egg produced by the brown practical system possessed by the National Livestock Science Institute. Artificial culture of fertilized eggs was carried out according to the method established in the National Livestock Science Laboratory. First, eggs 3g heavier than the selected fertilized eggs were selected by a surrogate egg, and the sharp parts of the eggs were cut to 3.5cm in diameter and used. Prior to virus microinjection, the fertilized egg was excavated and the fertilized egg was transferred to a surrogate egg, and a recombinant lentiviral having approximately 1 μl of human lactoferrin gene was injected using a capillary glass tube under a microscope. After the filling of the surrogate egg shell completely by using the prepared culture solution, it was sealed with a wrap and incubated for 2 days in an incubator of 90 ° every 11 minutes in an incubator having a temperature of 37.6 ° C. and a humidity of 65% -70%. After completion of the secondary culture for 3 days, the third surrogate egg shell was selected as a fresh egg about 25g heavier than the weight of the fertilized egg, and cut into the obtuse end with 4cm diameter, and then used as surrogate egg shell. The fertilized egg and its contents were transferred to the prepared surrogate egg shell, sealed with a wrap, and incubated for 15 days while incubating at 30 ° every 30 minutes in an incubator having a temperature of 37.6 ° C. and a humidity of 65% -70%. After incubation was incubated without eggs. Recombinant lentivirus containing human lactoferrin gene was microinjected into 539 fertilized eggs, and as a result of incubation, 24 candidate transgenic eggs successfully hatched, and 19 transgenic animals survived for more than a week after hatching. In the control group, which was subjected to artificial culture using the surrogate egg method without microinjection of virus, 27 chicks, which were approximately 43%, hatched normally in 63 fertilized eggs. It also shows that the fertilized egg culture system of chicken using surrogate egg was working normally. Overall fertilized embryo culture and candidate transgenic chick production results are shown in Table 1.

Table 1. Result of artificial culture of fertilized egg using surrogate egg method

Experimental group Stage-X embryos
(n) Survival of embryos during culture No. of hatched chicks
(%) No. of
1 week old birds (%) No. of twin
(%) 4 days
(%) 11 days (%) 19 days (%) Lactoferrin 539 478
(88.7) 129
(23.9) 86
(16.0) 24
(4.45) 19
(3.5) 8
(1.5) Control 63 - - - 27
(42.9) - -

3. Selection of the first generation candidate transgenic chicken (G0)

Since the first generation transgenic chicken (G0) produced by injecting recombinant virus into blastocyst stage embryos is produced in 100% mosaic form, the true transgenic chicken should be produced with second generation transgenic chicken (G1). It can be said that transgenic chickens have been produced. Due to this method characteristic, selection of the first generation transgenic chicken (G0) having a foreign gene carried in a recombinant virus in semen is an essential process. The semen analysis of males of candidate transgenic chickens produced by microinjecting human lactoferrin gene confirmed that the target gene, ie, human lactoferrin gene, exists in two semen (FIG. 5). Primers and PCR conditions used for the PCR verification are as follows.

OVP F (80): 5-ggt caa act tct gaa ggg aac ctg tg-3, LTF R (120): 5-cca ttg gaa gca ttt tgt ggc ctc g-3 primers and taq-polymerase (AccuPrime SuperMix II, PCR was performed under conditions of 94 ° C.-2 min, 94 ° C.-45 sec, 62 ° C.-30 sec, and 68 ° C.-20 sec. Individuals selected for the presence of the gene of interest in the semen are subject to re-validation, not to the promoter region of the expression vector, but to the 3-UTR region VectorCoA IRES R: 5-CAG CGG CTT CGG CCA GTA ACG TTA G-3, LTF F (1900). : PCR analysis was performed under the same conditions as the promoter region using 5-GAG AAA TGG ATC TGA CTG CCC GGA C-3 primers to confirm the presence of the target gene (FIG. 6). In addition, PCR amplification was indirectly confirmed that the amplified DNA fragments were specifically amplified by cleaving the amplified DNA fragments in the same form as the control group as a result of treating the amplified DNA fragments with EcoRI restriction enzymes (FIG. 6). ). In addition, the DNA fragments of the 3-UTR region and the promoter specifically amplified in FIGS. 5 and 6 were cloned and confirmed to be human lactoferrin genes (FIG. 7).

4. Selection of transgenic chicken (G1) with human lactoferrin gene

Two semen of transgenic chickens having the target gene in semen were artificially inseminated in normal females to produce second generation candidate transgenic chicks. Genomic DNA required for genetic analysis was extracted by collecting 5 μl of blood from the wing vein of the candidate transformed chicks. PCR genetic analysis was performed according to the established method (Fig. 8). PCR analysis showed positive results in 2 blood. Second-generation transgenic chickens (G1), first selected by PCR, were re-extracted for revalidation, extracted genomic DNA, treated with EcoRI restriction enzyme shown in FIG. 1, and then human lactoferrin gene was used as a probe. Southern blot analysis was performed (FIG. 9). PCR and Southern analysis showed the same results. The above results show that the complete transgenic chickens are produced instead of the mosaic form into which the human lactoferrin gene is introduced. Table 2 summarizes the production results of the first generation and second generation transgenic chickens. In short, semen was collected from two numbers of first-generation transgenic chickens (G0) and artificially inseminated to normal hens to produce more than 3000 fertilized eggs, and 1,900 candidate transgenic chickens were produced from them. Blood collection, genomic DNA isolation, and PCR analysis were performed on all individuals to finally select two transgenic chickens (G1).

Table 2. Second Generation Lactoferrin Transgenic Chicken Production Results

ID Ranunculus Incubation water (%) Sperm water (ml) Sperm Activity Positive entity (G1) A278 (G0) 1597 1200 (75) 26.4 x 10 ⁸ height One A279 (G0) 1514 667 (44) 9.2 x 10 ⁸ lowness One

1 is a schematic diagram of a lentiviral vector having a human lactoferrin gene.

Figure 2 shows the results of PCR amplification of the ovalbumin promoter (A) and human lactoferrin gene (B) of the chicken.

3 is a lentiviral vector having an ovalbumin promoter of chicken and a lactoferrin gene of human.

Figure 4 shows the sequence analysis. Red: Chicken ovalbumin promoter sequence, Blue: Human lactoferrin gene sequence, Black: Lentivirus vector sequence. GAATTC: EcoRI restriction site, ATG: start codon, TAA: stop codon.

Figure 5 shows the semen PCR analysis of the candidate transgenic chicken (promoter region). Pc: Positive control, Nc: Negative control.

Figure 6 shows the semen PCR analysis of the candidate transgenic chickens (3-UTR region). Pc: Positive control. Lane 1: before treatment with EcoRI restriction enzyme, lane 2: after treatment with EcoRI restriction enzyme.

Figure 7 shows the sequence analysis of the PCR amplified DNA fragments in the semen of the first generation transgenic chickens (G0). (A): promoter region, (b): 3-UTR region, red: ovalbumin promoter sequence, blue: human lactoferrin gene sequence, green: lentivirus vector sequence, and black: TA-cloning vector Base sequence. GAATTC: EcoRI restriction site, ATG: start codon, TAA: stop codon.

8 is a result of genomic DNA PCR analysis extracted from blood of the second generation transgenic chicken (G1). Pc: Positive control, Nc: negative control.

Figure 9 is a second generation transgenic chicken (G1) Southern analysis of the human lactoferrin gene. Lane 1, 2: Normal Chicken; lane 3, 4: lactoferrin transgenic chickens, P1, 2: positive control.

Claims (5)

A lentiviral vector combining the human lactoferrin gene and the promoter region of a 2 kb ovalbumin gene in chickens. Recombinant lentivirus produced in 293FT virus packaging cells using the lentivirus vector according to claim 1. In the artificial culture method of chicken fertilized egg, After digging the fertilized egg of the chicken and moving the contents to the surrogate eggshell, Method for artificial culture of chicken fertilized egg having a human lactoferrin gene comprising the step of culturing after injecting the recombinant lentiviral according to claim 2 to the transferred contents. The method of claim 3, wherein The fertilized egg is artificial culturing method of chicken fertilized egg having a human lactoferrin gene, characterized in that the embryonic stage of fertilization. The method according to claim 4 analyzes the semen of males of transgenic chickens and selects the presence of human lactoferrin gene. A method for producing a transgenic chicken having human lactoferrin gene, characterized in that the male semen selected above is artificially fertilized and hatched to a normal chicken female.

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